The conjugation of water-soluble polyalkylene oxides with therapeutic moieties such as proteins and polypeptides is known. See, for example, U.S. Pat. No. 4,179,337, the disclosure of which is hereby incorporated by reference. The '337 patent discloses that physiologically active polypeptides modified with PEG circulate for extended periods in vivo, and have reduced immunogenicity and antigenicity.
To conjugate polyalkylene oxides, the hydroxyl end-groups of the polymer must first be converted into reactive functional groups. This process is frequently referred to as “activation” and the product is called an “activated polyalkylene oxide.” Other polymers are similarly activated.
Amine terminated polymers such as PEG-NH2 are known. See Zalipsky et al. Eur. Polym. J. (1983) Vol. 19 No. 12., pp 1177-1183. They can be used “as is” for direct conjugation to COOH groups found on some biologically active compounds. More often, PEG-NH2 (or PEG-amine) is used as an intermediate which is further functionalized when other polymeric delivery systems are desired. For example, certain polymer-based drug delivery platform systems containing benzyl elimination systems, trimethyl lock systems, etc. can include PEG-NH2 as a key intermediate in the process of synthesis. See Greenwald et al. J. Med. Chem. Vol. 42, Sep. 9, 1999; No. 18, 3657-3667; Greenwald et al. J. Med. Chem. Jan. 29, 2004; Vol. 47, No. 3, 726-734; Greenwald et al. J. Med. Chem. Feb. 10, 2000; Vol. 43, No. 3, 475-487. The contents of each of the foregoing are hereby incorporated herein by reference.
PEG-amines are also useful for conjugation (via reductive amination) with biologically active small molecules and polypeptides having available aldehyde groups. See also Nektar Advanced PEGylation catalog 2005-2006, page 24, the contents of which are incorporated herein by reference.
In the past, it was generally known that PEG-amines could be prepared by preparing the PEG-halide, mesylate or tosylate from PEG-OH and thereafter performing a nucleophilic displacement reaction with aqueous ammonia (Hoffmann Reaction), sodium azide or potassium phthalimide (Gabriel Reagent). The reaction of the PEG-halide with the ammonia forms the PEG-amine directly. More importantly, a major disadvantage is that a significant percentage of PEG-halide becomes hydrolyzed to form PEG-OH during the concentrated aqueous ammonia treatment. This is a particular concern when forming higher molecular weight PEG-amines. The higher molecular weight PEG, the more PEG-OH is formed. For example, in the case of PEG5,000 the amount is about 5% and with higher molecular weight PEG such as PEG40,000 the amount can be up to 20%. Consequently, the purity of the desired end product can decrease considerably.
Even when PEG-azide is used as the intermediate to make the PEG-amine, certain shortcomings have been observed when metal catalyzed hydrogenation is used. Furthermore, reaction with potassium phthalimide provides a basically protected amine that is deprotected with hydrazine in ethanol under reflux. This too is associated with drawbacks. The harsh conditions required for removal of the phthaloyl group and the need for intensive purification of the final product add significantly to the cost of the desired product.
In view of the foregoing, it would be desirable to provide improved methods for preparing PEG-amines and related polymers having terminal amines which address the shortcomings and drawbacks of the prior art. The present invention addresses this need.